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Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs

Nature Nanotechnology volume 17, pages 1120–1126 (2022)Cite this article

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Abstract

CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence that activates Cas enzymes to cleave reporter molecules. Currently, most CRISPR-based diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here we show the combination of a CRISPR–Cas-based reaction with a nanozyme-linked immunosorbent assay, which allows for the quantitative and colorimetric readout of Cas13-mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate that CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilize this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme will serve as a universally applicable signal catalyst for CRISPR-based diagnostics, which will expand the spectrum of targets for preamplification-free, quantitative detection.

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Fig. 1: CrisprZyme assay scheme.
Fig. 2: Pt@Au functionalized with streptavidin shows the best NLISA performance.
Fig. 3: CrisprZyme detects synthetic RNA down to picomolar concentration.
Fig. 4: CrisprZyme expands the dynamic range of Cas13-based diagnostics enabling the quantitative sensing of different non-coding RNA species.
Fig. 5: Quantification of different ncRNA species from cell culture and human plasma or tissue.

Data availability

Research data are available online at https://doi.org/10.5281/zenodo.6553774.

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Acknowledgements

We acknowledge use of the characterization facilities at the Harvey Flower Electron Microscopy Suite (Department of Materials, Imperial College London). We thank the PCBN for providing patient samples. This work was supported by the Department of Defense Prostate Cancer Research Program, US Department of Defense Award Nos W81XWH-18-2-0013, W81XWH-18-2-0015, W81XWH-18-2-0016, W81XWH-18-2-0017, W81XWH-18-2-0018 and W81XWH-18-2-0019 PCRP PCBN. M.B. and M.M.S. acknowledge funding from the British Heart Foundation (grant no. RE/18/4/34215) and the EPSRC IRC in Early Warning Sensing Systems for Infectious Diseases (i-sense) Mobility Fund (grant no. EP/K031953/1). M.M.K. was supported by the German Academy of Sciences, Leopoldina (grant no. LPDS 2018-01) and the Emmy Noether Programme (grant no. KA5060/1-1). M.M.K. is a participant in the BIH Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health at Charité (BIH). C.A. was supported by the Singapore A*STAR National Science Scholarship fellowship. A.J.S. was supported by the MD Research Stipend of the BIH. H.K. and M.M.S. acknowledge funding from the Research Council of Norway through its Centres of Excellence scheme (grant no. 262613). X.T. is supported by an American Gastroenterological Association Takeda Pharmaceuticals Research Scholar Award in Inflammatory Bowel Disease and the Wyss Institute. J.J.C. was supported by the Paul G. Allen Frontiers Group and the Wyss Institute. M.M.S. acknowledges funding from the EPSRC IRC in Agile Early Warning Sensing Systems for Infectious Diseases and Antimicrobial Resistance (i-sense2) (grant no. EP/R00529X/1), the Royal Academy of Engineering Chair in Emerging Technologies award (no. CiET2021\94) and the Rosetrees Trust. We thank A. Schütz and the team of the Protein Production & Characterization Technology Platform of the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany (https://www.mdc-berlin.de/protein-production-characterization) for producing the LbuCas13a protein.

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Author notes

  1. These authors contributed equally: Marta Broto, Michael M. Kaminski, Christopher Adrianus.

Authors and Affiliations

  1. Department of Materials, Department of Bioengineering, and Institute of Biomedical Engineering, Imperial College London, London, UK

    Marta Broto, Christopher Adrianus, Nayoung Kim, Schan Dissanayake-Perera, Hyemin Kim & Molly M. Stevens

  2. Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

    Michael M. Kaminski, Robert Greensmith & Alexander J. Schubert

  3. Department of Nephrology and Medical Intensive Care, Charité Universitätsmedizin Berlin, Berlin, Germany

    Michael M. Kaminski, Robert Greensmith & Alexander J. Schubert

  4. Berlin Institute of Health, Berlin, Germany

    Michael M. Kaminski & Alexander J. Schubert

  5. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Xiao Tan & James J. Collins

  6. Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA

    Xiao Tan

  7. Harvard Medical School, Boston, MA, USA

    Xiao Tan & Anand S. Dighe

  8. Institute for Medical Engineering and Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Xiao Tan & James J. Collins

  9. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Anand S. Dighe

  10. Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    James J. Collins

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  1. Marta Broto
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Contributions

M.B., M.M.K., J.J.C. and M.M.S. conceived and designed the research. M.B., M.M.K. and C.A. carried out all the experiments and analysed the data. N.K. performed the LFA experiments, TEM, STEM and EDX imaging and analyses. R.G. and A.J.S. performed RT–RPA, assisted in the design and production of gRNAs and in the optimization of LbuCas13a detection. S.D.-P. optimized and printed LFA strips. X.T. and A.S.D. collected patient samples and edited the manuscript. H.K. performed cell differentiation. M.B., M.M.K., J.J.C. and M.M.S. wrote the manuscript with feedback from all the authors.

Corresponding authors

Correspondence to James J. Collins or Molly M. Stevens.

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Competing interests

M.M.S., M.B., C.A. and S.D.-P. have filed a patent application (2110729.7) covering the techniques and assay design as described in the manuscript. J.J.C. is a co-founder and director of Sherlock Biosciences. The other authors declare no competing interests.

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Supplementary equation (1), Figs. 1–10 and Tables 1–5.

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Broto, M., Kaminski, M.M., Adrianus, C. et al. Nanozyme-catalysed CRISPR assay for preamplification-free detection of non-coding RNAs. Nat. Nanotechnol. 17, 1120–1126 (2022). https://doi.org/10.1038/s41565-022-01179-0

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